Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Actinomyces israelii, was investigated using a susceptibility assay. Human gingival fibroblasts were treated with NO-releasing compounds at the minimum concentrations required for bacterial growth and cytotoxicity was evaluated using the MTT cell proliferation assay. Our results suggest that NO-releasing compounds can be used topically to treat both gram-negative and gram-positive periodontal pathogens. Comparison of the antimicrobial activity and cytotoxicity assay results between the NO-releasing compounds revealed that an NO donor comprising a macromolecule without surface charge, a lower instantaneous NO concentration, and an adequate supply of NO were associated with a strong bactericidal effect and low cytotoxicity. NO-releasing compounds with these properties may be suitable for treatment of periodontitis.This study describes the successful synthesis of nitric oxide (NO)-releasing compounds with biodegradable and injectable properties and demonstrates that the kinetics of NO release vary according to the type of NO donor. The antimicrobial activity of NO-releasing compounds against three common periodontal pathogens, i.e.,  Antibacterial agents can be administered systemically or topically for control of infection in periodontal tissue. Systemic administration of antibiotics has been proven to be effective in suppressing progression of periodontitis . HoweverBiodegradable and injectable materials are suitable for topical delivery of antibiotics. Non-biodegradable materials should be removed after the antibiotic is released and may cause irritation and inflammation at the treated site. A biodegradable vehicle has the advantage of keeping patient visits to a minimum. Injection by syringe is a simple procedure for delivery of antibiotic agents and ensures compliance. The procedure requires standard and inexpensive devices, and the antibiotic can be applied rapidly and easily without pain or discomfort to the patient .Nitric oxide (NO), a gaseous reactive free radical species produced in mammalian cells, plays a crucial role in the immune response to pathogens  and is i- functional group are able to generate two molecules of NO from each moiety, so have been widely used to construct NO delivery systems. These NO donors can be easily functionalized onto any material containing secondary amine groups. NO-releasing compounds containing diazeniumdiolates have now been synthesized using various scaffold molecules, and their antimicrobial activity and cytotoxicity have been investigated. Interestingly, it was found that a macromolecular-based NO delivery system had greater antibacterial activity than a delivery system containing a small-molecule NO donor m (maximum instantaneous concentration of NO released), tm (time required to reach [NO]m), and t1/2 . For the bactericidal and cytotoxicity assays, the NO compounds were brought into contact with bacteria and cells, respectively, 10 min after they were mixed with solution. The kinetics of NO release were recorded at 0 and 10 min after the initial NO release response.The amount of NO released was measured using a Sievers 280i Nitric Oxide Analyzer , which measures chemiluminescence. The NONOate-containing materials were added to Dulbecco’s phosphate-buffered saline, and the release profile was obtained. The DETA-NO release profile was analyzed up to its detection limit (1 pmol). NO release was evaluated by characterizing multiple NO parameters, including [NO]Aggregatibacter actinomycetemcomitans (ATCC 43718), Porphyromonas gingivalis (ATCC 33277), and Actinomyces israelii (ATCC 12102) were purchased from the American Type Culture Collection . For verification of decontamination, P. gingivalis was cultured in trypticase soy agar supplemented with 5% sheep blood, hemin (1 μg/ml), and vitamin K (0.2 μg/ml), and A. actinomycetemcomitans and A. israelii were grown in brain heart infusion (BHI) agar at 37°C under anaerobic conditions . A single colony of each type of bacterium was then cultivated with the appropriate medium. For the susceptibility assay, P. gingivalis was cultivated in BHI broth containing hemin (1 μg/ml) and vitamin K (0.2 μg/ml) and A. actinomycetemcomitans and A. israelii were cultivated in BHI broth at 37°C under anaerobic conditions. The bacteria were then counted using a bacteria counting chamber  and the concentration of each type of bacteria was adjusted to 1 × 107 cells/ml by addition of fresh medium.P. gingivalis or BHI broth for A. actinomycetemcomitans and A. israelii were dispensed into the wells of the plate. A solution containing Py-NO, DETA-NO, or FBN dissolved in sterilized, distilled H2O was added to the 12th column of the 96-well plate and serially diluted 2-fold progressing to the second column using a multi-channel micropipette. The first column of each well contained broth only and served as a negative control. Finally, 20-μl bacterial suspensions (containing 1 × 105 cells) were inoculated into each well. The plates were then incubated at 37°C for 36 h under anaerobic conditions. Bacterial growth was measured by optical density at a wavelength of 660 nm using a microplate reader . A 96-well plate containing medium with dilution of NO-releasing compounds and inoculation of periodontal pathogens was incubated for evaluation of the minimum bactericidal concentration (MBC). A 50-μl aliquot of liquid from each well was inoculated and spread onto BHI agar for A. actinomycetemcomitans and A. israelii or onto trypticase soy agar containing sheep blood, hemin, and vitamin K for P. gingivalis. The plates were then incubated at 37°C for 3 days (A. actinomycetemcomitans and A. israelii) or 5 days  under anaerobic atmospheric conditions. The lowest concentration that revealed no visible bacterial growth after subculturing was taken to be the MBC.The antimicrobial activity of the NO-releasing compounds against periodontal pathogens was evaluated using a susceptibility assay according to the methods of the Clinical and Laboratory Standards Institute . A minim2. The cells were seeded with medium (100 μl) in a 96-well plate (SPL Life Sciences) at a density of 1 × 104 cells/well and incubated at 37°C for 24 h. Solutions (100 μl) of control medium and NO-releasing medium prepared in Dulbecco’s modified Eagle’s medium by the same method as that used for the bactericidal assay were added to the cells and incubated at 37°C for 24 h. Next, 20 μl of a 5 mg/ml solution of MTT was added to each well and incubated for 4 h in the dark; 100 μl of medium was then aspirated carefully from each well and 100 μl of dimethyl sulfoxide was added to each well to dissolve the formazan precipitate. Absorption was measured at a wavelength of 570 nm using a VERSAmax tunable microplate reader (Molecular Devices). The viability of the cells in each well was calculated by comparing it with that of an untreated control (100%).HGF-1 (ATCC #CRL-2014) cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and incubated at 37°C under humidified conditions in 5% COU test, using SPSS version 23 software . A p-value <0.05 was considered statistically significant.Differences between cell and bacterial samples treated or not treated with the NO-releasing compounds were assessed for statistical significance using the Kruskal-Wallis test and the Mann-Whitney m, time required to reach the half-life of NO release, number of moles of NO released per mg from 10 min to 24 h, and the instantaneous concentration of NO released at 10 min were recorded for each compound . To inhibit the growth of A. actinomycetemcomitans, the concentrations of NO-releasing compounds required were 0.39 mM for DETA-NO, 1.5 mM for F68-BPEI-NO, 1 mM for F68-BPEI, and 12.5 mM for Py-NO , P. gingivalis (gram-negative), and A. israelii (gram-positive). The cytotoxic effects of the NO-releasing compounds on HGF-1 cells at the MBC were measured to evaluate the therapeutic potential of these agents.This study examined the potential suitability of NO-releasing compounds for use in clinical practice as antibacterial agents against periodontal pathogens. Py and DETA are organic compounds with monomolecular structures that are soluble to water and polar organic solvents ,17. AlthLike in previous studies, the kinetics of NO release varied depending on the NO delivery system used ,21, althA. actinomycetemcomitans and P. gingivalis are gram-negative periodontal pathogens that were susceptible to NO. This finding corresponds to the results of previous studies [ studies ,20. Py-N studies ,20. Furt studies . TherefoBactericidal efficacy depends on surface charge as well as the kinetics of NO release; bacterial cell walls are negatively charged, and positively charged macromolecules can interact with and disrupt bacterial cell membranes via electrostatic interaction . F68-BPEA. israelii, a gram-positive periodontal pathogen and found results similar to those of the bactericidal assays against gram-negative periodontal pathogens. A. israelii is only one of the strains of bacteria that cause gingivitis; Actinomyces spp. are also significant pathogens in the etiology of medication-related osteonecrosis of the jaw (MRONJ) [We evaluated the bactericidal efficiency of NO against  (MRONJ) ,28. Ther (MRONJ)  and an a (MRONJ) , and als (MRONJ) .F68-BPEI becomes strongly positive when secondary amines are attached, causing a non-specific ionic interaction with negatively charged bacterial membranes and zwitterionic cell membranes. Therefore, F68-BPEI is highly cytotoxic and also inhibits growth of periodontal pathogens. Interestingly, in the present study, the cytotoxic effect of F68-BPEI-NO was improved after addition of NONOates. The positive charge of F68-BPEI (20.88 mV) was reduced when attached to NONOates, and F68-BPEI-NO had a surface charge that was close to zero (0.43 mV). NO was added at a relatively low concentration, suggesting that inducing zero surface charge by addition of NO may have a positive effect on cells. Although a positive surface charge can improve the bactericidal effect, a lack of surface charge seems to be more beneficial for the therapeutic use of NO with respect to cytotoxicity. Py-NO was more cytotoxic than DETA-NO in our study, which confirms that an instantaneous NO concentration is more important for increasing cytotoxicity than the total amount of NO released ,31. TherStaphylococcus aureus [Eradication of microorganisms from the periodontal pocket is the most important step in treating periodontitis. The limited effects of mouth rinsing and irrigation have prompted research on alternative antimicrobial delivery systems. Advances in drug delivery technology have allowed development of controlled-release drug formulations. The requirements for a drug delivery system to treat periodontitis include maintenance of an effective drug concentration for an adequate duration at the site of infection and minimal or no side effects in periodontal tissue . Variouss aureus . HoweverF68-BPEI-NO may be a prepotent candidate for a topical agent in the treatment of periodontal disease because it demonstrated sufficient bactericidal effects on periodontal pathogens, with minimum cytotoxicity to HGF-1 cells. NO donors composed of a macromolecule without surface charge, a lower instantaneous NO concentration, and sufficient total amount of NO had a strong bactericidal effect and low cytotoxicity.S1 FigIn comparison to py-NO and DETA-NO, pyrrolidine and DETA showed antimicrobial activity at a concentration of 10-fold or more, and their antimicrobial effect was attributable to release of NO.(TIFF)Click here for additional data file.


Clostridium difficile (CD) infection as a consequence of gut dysbiosis. Currently available treatments of IBD are either not very effective or have adverse effects. Pyungwi-san (PWS), a traditional Chinese herbal formulation, has long been used to treat gastrointestinal disorders. The present study was conducted to investigate the efficacy of PWS against dextran sulfate sodium (DSS) + CD-induced IBD in mice. The animals received DSS in drinking water for seven days to produce DSS-induced acute colitis. In the DSS + CD group, the DSS-fed animals were orally administered with CD spores twice during the DSS treatment period. We observed that exposure of DSS + CD-treated animals to PWS significantly decreased the disease activity index; prevented the shortening of colonic length and increases in spleen size and weight; restored colonic histological parameters by significantly increasing mucus thickness, crypt depth, and goblet cell numbers; protected the tight junction proteins; improved the profiles of pro-inflammatory and anti-inflammatory cytokines; and normalized the abundance ratio of the Firmicutes/Bacteroidetes in the gut. Thus, PWS exerted a number of protective effects on DSS + CD-induced colitis, which might be mediated via restoration of a balance in gut microbial communities.Several lines of evidence indicate that inflammatory bowel disease (IBD) is associated with  Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn’s disease, is a chronic inflammatory intestinal condition manifested by abdominal pain, weight loss, diarrhea, rectal bleeding, and diffuse mucosal inflammation ,2. AccumA characteristic feature of patients suffering from IBD is the dysfunction and deformity of the intestinal epithelial tight junction (TJ), which is manifested by low-level expression of intestinal epithelial TJ-related proteins and/or their abnormal distribution ,5. This Clostridium difficile (CD) periods, claudin-1 and occludin show significant decreases in their expression levels at the protein and mRNA levels in the colonic mucosa, which has been associated with damage of the intestinal epithelial barrier and a concomitant increase in permeability of the intestinal epithelium [Claudins and occludin are the two key proteins in the TJ . The claithelium ,12.It has been reported that interleukin (IL)-1β, a pro-inflammatory cytokine, is likely to have a role in the pathogenesis of IBD . A previClostridium difficile, an anaerobic gram-positive, spore-forming, toxin-producing bacillus that causes intestinal diseases ranging from mild diarrheal illness to severe colitis. Patients with IBD are at higher risk of susceptibility to C. difficile infection (CDI), have worse outcomes of CDI including increased rates of colectomy and death, and exhibit a higher recurrence rate [Gut microbes are considered to be key factors in intestinal inflammation in IBD and a number of studies have suggested that dysbiosis occurs in IBD . An accunce rate .Pyungwi-san (PWS) decoction, a mixed herbal formulation composed of the extract of six herbs, has been used in clinics for thousands of years in China owing to its anti-oxidative and anti-inflammatory activities [Tai ping hui min he ji ju fang) [Pyung  signifies calming down of all the unbalanced things. Wi (stomach) does not only represent the stomach organ; rather it defines the entire digestive system that removes obstruction and stagnation by elimination through the intestine [Citri Pericarpiumpeel has traditionally been used in Asia as a medicine for the improvement of asthma and adverse bronchial conditions or blood and cardiac circulation [glycyrrhizin, a bioactive triterpene saponin present in Glycyrrhiza uralensis of PWS formulatiion, causes strong attenuation of inflammatory responses induced by Toll like receptor (TLR) 3 and TLR 4 ligands [magnolol, the active compound of the Magnoliae Cortex, another component of PWS, has been shown to possess anti-inflammatory activities [Rhizoma Atractylodis Macrocephalae (RAM), a herb that is also a constituent of PWS, and its main compounds are known to exert various pharmaceutical impacts on experimental models, including anti-cancer [Thus far, the therapeutic strategies adopted for combating IBD encompass treatment of colitis via administration of anti-inflammatory or immunosuppressive drugs, antibiotics, and surgery which aims to regulate immune cell-derived cytokine production. However, undesirable side effects such as allergies, fever, cramps, lymphoma, and diabetes limit the long-term use of these therapies. Thus, a lot of research has been undertaken to find alternative therapeutic strategies for IBD treatment, strategies that will be efficacious on the one hand and devoid of undesirable side effects on the other ,21. Thertivities . The desju fang) . In tradntestine . On the ntestine ,28,29,30culation . It has  ligands . This mo ligands . Moreovetivities ,35. Addii-cancer ,37, antii-cancer , anti-uli-cancer , anti-oxi-cancer , anti-ini-cancer , and anti-cancer  effects.C. difficile-induced mouse models of IBD.Accumulating evidence indicates the usefulness of PWS in the treatment of common gastrointestinal disorders, such as gastritis, esophageal reflux, gastric or duodenal ulcers, and acute or chronic enteritis . It has No significant difference in body weight was detected among the study groups on day 0 of the experimental period B,C. Withp < 0.0001) at both days 6 and 7 of the experimental period However, treatment of the DSS + CD group with PWS significantly decreased the disease severity at both of these times (p < 0.001).As expected, no sign of disease activity was seen in the animals of the normal group throughout the experimental period D,E. In cTo further assess the protective effect of PWS against DSS + CD-induced adversity, tissue parameters from samples collected from animals sacrificed animals on day 7 were analyzed. Colonic length and cecum weight were significantly lower in both the DSS and DSS + CD groups compared with those of the normal group A–C. HoweTo determine whether PWS treatment protects against histological damage induced by DSS or DSS + CD treatments, we performed hematoxylin and eosin (H&E) and Alcian blue (AB)-staining of the colonic tissue sections (p < 0.0001) than by DSS alone (p < 0.001) (p < 0.0001) than the former one (p < 0.001) (p < 0.001) in the AB-stained area was observed in the DSS + CD + PWS group, indicating a notable response to co-treatment with PWS. In support of the above results, a significant reduction (p < 0.0001) in crypt depth and quantity of goblet cells was observed upon treatment with both DSS and DSS + CD (p < 0.001) and the goblet cell number was significantly increased (p < 0.01) in the DSS + CD mice co-treated with PWS.Histological evaluation of the AB-stained colonic tissue was performed to investigate the structural integrity of the intestinal mucosa. As expected, exposure of mice to DSS or DSS + CD resulted in marked destruction of the colonic epithelial architecture A. Specif< 0.001) B. Howeve< 0.001) A,C. NeveDSS + CD A,D,E. HoTo evaluate the anti-inflammatory effects of PWS on DSS + CD-induced colitis, we focused on the pro-inflammatory cytokine IL-1β, and the anti-inflammatory cytokines IL-10 and TGF-β. The Western blot analyses of colonic tissue lysates revealed a higher expression of IL-1β and a lower expression of IL-10 and TGF-β in both the DSS and DSS + CD groups compared with those of the normal group . HoweverTo get an indirect measure of the extent to which the TJ is disrupted in response to DSS- and DSS + CD-induced colitis, the colonic expression of TJ markers, namely occludin and claudin-1, was analyzed. Tissue levels of claudin-1 were decreased in response to both DSS and DSS + CD treatments . SimilarBacteroidetes was lower in the DSS + CD group than that in both the DSS and normal groups, whereas the relative abundance of Firmicutes was lower in both the DSS and DSS + CD groups than in the normal group, although the differences were not statistically significant. Notably, the ratio between these two phyla (Firmicutes/Bacteroidetes) was significantly decreased in the DSS + CD-treated mice co-treated with PWS  was higher in both the DSS and DSS + CD groups, but the difference was significant only in the latter group. The relative abundance of E. coli (S) was lower in the DSS + CD + PWS group than that in both DSS and DSS + CD groups. The relative abundance of Clostridium cluster IV (S) was markedly lower in all the treated groups, including the DSS + CD + PWS group, compared with that in the normal group. The relative abundance of Akkermansia muciniphila (S) was significantly higher in the DSS group than in the normal group. Co-treatment of DSS + CD-treated mice with PWS resulted in a decrease in the abundance of A. muciniphila (S), although the difference was not statistically significant (p = 0.0695). The rRNA-based terminal fragment length polymorphism (T-RFLP)-based analyses of stool samples collected at the termination of the experimental period revealed a marked variation in the distribution of gut microbial population among the experimental groups A. At thewith PWS B. At theC. difficile, a bacterial strain that is known to be a major cause of hospital-acquired infection and is responsible for the development of symptoms ranging from diarrhea to severe colitis, sepsis, and ultimately death. Consistent with these findings, there is an accumulation of evidence indicating an increasing incidence of CDI and associated pathogenicity in IBD patients [C. difficile and IBD is mediated by a series of events including recurrent hospital admissions, which are a known risk factor for acquisition of C. difficile and CDI; drug administration to patients with IBD, especially antibiotics that destabilize intestinal microbial communities, thus permitting C. difficile colonization and adherence; and nutritional status deterioration, which promotes C. difficile infection.Until now, IBD has been considered an outcome of a disturbed mucosal immune response to multiple factors that lead to chronic intestinal inflammation and tissue destruction ,45. Notapatients ,47. It hC. difficile and IBD may be mediated by a number of factors, including recurrent hospital admissions, a known risk factor for acquisition of C. difficile and CDI; drug administration to IBD patients, especially antibiotics that destabilize the intestinal microbial communities, thus permitting C. difficile colonization and adherence; immune system and nutritional status alteration; and genetic predisposition [Furthermore, it has been suggested that the association between position .C. difficile secretes two types of potent exotoxins: toxin A (“enterotoxin”) and toxin B (“cytotoxin”), which bind to receptors on intestinal epithelial cells. These secretions activate a cascade of events and trigger the expression of pro-inflammatory cytokines and leukotrienes, inducing apoptosis of gut epithelial cells and increasing the permeability of the intestinal mucosa, which, in turn, can have a role in the pathogenesis of IBD [Following its colonization in the intestine of IBD patients, s of IBD .C. difficile infection, has not been previously evaluated. Thus, in the present study, we investigated the effects of PWS in a mouse model of IBD. Our results revealed that PWS treatment can exert a potentially beneficial effect on DSS + CD-induced colitis, as reflected by the observed changes in clinical symptoms and pathophysiology such as improvement in stool-types disease activity index (DAI) scores and amelioration of shortening of colonic length. These results are consistent with the outcomes reported in previous clinical studies in which PWS treatment produced a significant improvement in clinical symptoms in up to 93.0% of patients suffering from ulcerative colitis [For the treatment of IBD, a number of studies have investigated the applicability of traditional oriental medicines as an alternative to commonly used Western drugs such as anti-bacterial, anti-inflammatory, and immunosuppressive agents , which c colitis . Our stu colitis ,53. AddiIt has been suggested that pro-inflammatory cytokines have a vital role in the course of colitis in both mice and humans . An accuThe epithelium in the gut has an important role in the pathogenesis of IBD. Intestinal permeability is mainly maintained by TJs, which regulate the functioning of the epithelial barrier . IncreasC. difficile colonization [The integrity of the intestinal barrier also depends on the status of the intestinal immune system, which is largely influenced by gut microbial homeostasis. A healthy gut microbial community has a vital role in the protection of the host against the invasion and systemic dissemination of pathogenic microorganisms . An imbanization .Bacteroidetes was lower in the DSS + CD group than in both the DSS and normal groups. Although the differences were insignificant, a previous study showed that phylum distribution in IBD patients harbored significantly fewer Bacteroidetes than that in healthy subjects [C. difficile and abundance of Bacteroidetes [Firmicutes/Bacteroidetes phyla was higher in the DSS + CD group than in the normal group, supporting the findings of a previous study [In our study, the community structure of the gut microbiota was analyzed using T-RFLP, revealing that, at the phylum level, the population of subjects . Furtherroidetes . In addius study . HoweverE. coli abundance in response to treatment with DSS or DSS + CD, although the chance was significant only in the latter treatment. Several reports have revealed that the relative abundance of E. coli increases as a result of inflammation of the intestinal tract [E. coli [E. coli was observed to be lower in the DSS + CD + PWS group than in both the DSS and DSS + CD groups. In the present study, Clostridium cluster IV species showed a lower relative abundance in all treatment groups compared with the normal group, in support of the results in an earlier report [A. muciniphila (S), a mucin-degrading bacterium living in the mucus layer of the intestinal tract, has been documented in IBD patients [A. muciniphila (S) in mice treated with DSS, in agreement with the results of a previous study [A. muciniphila. In one of our previous studies, Rhizoma Atractylodis Macrocephalae, an active constituent of PWS, was shown to have a beneficial effect on the relative abundance of gut microbiota in rats treated with lipopolysaccharide [Atractylodes macrocephala was observed to significantly improve a disordered intestinal flora in rats; on that basis, it was surmised that this compound may be useful as an oral adjuvant in the regulation of intestinal flora [Z. officinale, another constituent of PWS, can modulate the composition of the gut microbiota, resulting in beneficial effects against a number of diseases including inflammation [At the species level, we observed an enrichment in al tract ,74,75,76[E. coli . Notablyr report . A shiftpatients ,78. Our us study . Howeverccharide . In agreal flora . A previammation . In agreSeven-week-old male C57BL/6 mice (ethics project identification code: 2017-06165) were purchased from Daehan Biolink Co. Ltd. . The animals were kept in a 12 h light/dark cycle under a constant temperature of 20 ± 3 °C with free access to autoclaved distilled water and standard chow. The animals were maintained under these conditions for one week to become acclimated. All procedures for animal experiments were performed in accordance with the principles outlined in the “Guide for the Care and Use of Laboratory Animals”  and approved by the “Institutional Animal Care and Use Committee” of Dongguk University.C. difficile (KCTC 5009) was purchased from the Korean Collection for Type Cultures . Culturing and strain typing of this bacterial strain were performed as previously reported [C. difficile was incubated in a taurocholate–cefoxitin–cycloserine–fructose agar (TCCFA) plate for 48 h at 37 °C in an anaerobic chamber . Eight single colonies from each plate were picked using sterile inoculating loops that were struck in quadrants on agar plates containing brain heart infusion medium supplemented with yeast extract  l-cysteine, 15 g agar per liter). Cultures were grown under anaerobic conditions at 37 °C for 5–7 days. Sporulation was induced on media containing BHIS and SMC , at 70% SMC and 30% BHIS  [g and were washed with ice-cold PBS five times after isolation. The spores were then incubated at 70 °C for 20 min to ensure that no residual vegetative cells survived. Viable spores were counted by plating as colony forming units (CFU)/mL on TCCFA to determine the challenge dose [reported . Brieflyr liter) . Spores nge dose .Atractylodes lancea, bark of Magnolia officinalis, Citrus reticulata Blanco, Glycyrrhiza uralensis, Zingiber officinalis, and Ziziphus jujube Mill. at a ratio of 29:14:20:9:14:14 ; (2) DSS (n = 12); (3) DSS + CD (n = 12); (4) DSS + CD + PWS (n = 13).Forty-nine male mice  were randomly assigned to one of four groups: (1) Normal (w/v) DSS  in drinking water for seven days (day 0 to day 6) in order to produce DSS-induced acute colitis in the animals. In the DSS + CD group, the DSS-fed animals were orally administered with 107 CFU of C. difficile spores twice over the period of treatment (days 0 and 3). In the DSS + CD + PWS group, the DSS + CD-treated mice received PWS (669.1 mg/kg/day) via oral gavage until the end of the experimental period (days 2 to day 7). During that same period, the mice in the normal, DSS, and DSS + CD groups were orally administered with the same volume of PBS without PWS.Prior to treatment, all mice were provided with free access to chow and distilled water as drinking water for two days . The entire colon (from cecum to anus) and the spleen were removed and their lengths were measured. The tissues were then washed with PBS (pH 7.4), dried, and weighed. The cecum was then excised and the colon length and weight were determined. The colon was cut into slices and some of them were immediately submerged in RNAlater™ Stabilization Solution  in sterile tubes followed by snap freezing in liquid nitrogen and storage at −80 °C for future RNA analysis. Some of the other colonic fragments were also rapidly frozen in liquid nitrogen and stored at −80 °C for future protein analysis by Western blotting. The remaining colonic tissue slices were fixed in 4% paraformaldehyde overnight and embedded in paraffin blocks for histological processing. In order to proceed for hematoxylin and eosin (H&E) staining, the tissues were sectioned to 5 μm thickness using a microtome  and placed on positively charged glass slides , followed by deparaffinization with xylene  and dehydration through an increasing ethanol concentration gradient. The tissue sections were stained with hematoxylin solution  and eosinY solution (0.5%) alcoholic  and then mounted on cover slips. The stained tissues were examined under an inverted light microscope  at 100×, 200×, and 400× magnifications. Microscopic images were captured using a digital camera . For Alcian blue (AB)-staining , the blo® Fast DNA Stool Mini Kit  per the kit manufacturer’s instructions. A previous study reported that DSS contamination of colonic tissue could inhibit the amplification of genomic DNA [Bacterial DNA was extracted from fresh stool samples that were collected on the last day of the treatment schedule; extraction was performed using the QIAampomic DNA . To avoiomic DNA . The extracted DNA solution was incubated on ice for 2 h with a 0.1 volume of 8 M LiCl  prepared in DNase/RNase-free water . The mixture was then centrifuged at 13,000 r/min for 30 min at 4 °C. The supernatant was discarded and the DNA pellet dissolved in 200 μL DNase/RNase-free water. That procedure was repeated once and the final obtained DNA pellet was incubated at −20 °C for 30 min, after which a 0.1 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of absolute ethanol were added. The mixture was centrifuged at 13,000 r/min for 30 min at 4 °C. The supernatant was discarded and the DNA pellet was washed with 70% ethanol and centrifuged at 13,000 r/min for 30 min at 4 °C. The supernatant was discarded and the DNA pellet dried and dissolved in 10–50 μL of DNase/RNase-free water.The purified bacterial DNA from feces was amplified via polymerase chain reaction (PCR) using an AccuPower PCR PreMix kit  and broad-ranged primers: 27F-FAM (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) . The conditions for PCR amplification reactions were as follows: initial denaturation for 3 min at 94 °C; 30 cycles of amplification encompassing denaturation at 94 °C for 1 min, annealing at 53 °C for 45 s, and elongation at 72 °C for 2 min followed by a final extension at 72 °C for 10 min. The PCR products were purified using a PCR purification kit  according to the kit manufacturer’s protocol, and the purified products were subjected to digestion with restriction enzyme Hap II . Finally, the digested DNA products were submitted to Macrogen Korea  for T-RFLP-based analysis of the microbial population using a 3730XL DNA analyzer . The T-RFLP electropherograms were visualized using GeneScan 3.7 software  and analyzed using XLStat software .® BCA protein assay kit  according to the kit manufacturer’s instructions. Forty micrograms of proteins were resolved by SDS gel electrophoresis on 15% acrylamide–bisacrylamide gels using 80–100 V for 80 min and then transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes  using a Mini Trans-Blot® electrophoretic transfer cell device . The membranes were then blocked with Tris-buffered saline  containing 0.1% Tween 20 (TBST) and 5% skim milk  for 1 h. Following this, the membranes were incubated overnight with the appropriate primary antibody at 4 °C as follows: anti-IL-10 , anti-TGF-β1 , anti-IL-1β , anti-occludin , or anti-claudin-1 . The immunoblots were then washed three times in TBST and incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit Immunoglobulin G (IgG) secondary antibodies  depending on the origin of the primary antibody. The housekeeping protein β-actin was used as a loading control for the samples. The immunoreactive bands were traced on a Las3000 imaging system  using Supex Enhanced chemiluminescence (ECL) reagent . Subsequently, the membranes were stripped in a buffer (62.5 mM Tris-HCl (pH 6.7) containing 2% SDS and 100 mM β-mercaptoethanol) and re-probed with anti-β-actin antibody  and processed identically as described above.Stored colonic tissues were homogenized on ice in RIPA buffer  containing cOmplete™, Mini, EDTA-free protease inhibitor , and the Xpert phosphatase inhibitor cocktail . The homogenates were centrifuged at 13,000 r/min for 15 min at 4 °C, after which the supernatants were collected. The protein concentrations of the homogenates were measured using a Piercep-values were determined using a two-tailed, unpaired Student’s t-test, or a repeated-measures analysis of variance (ANOVA) with Tukey’s correction for multiple comparisons.All data are expressed as means ± standard error (S.D.). The C. difficile-induced IBD in mice, as evidenced by the prevention of diarrhea, shortening of colon length, decrease in spleen size and weight, protection of TJ proteins, and improvement in the profiles of pro-inflammatory and anti-inflammatory cytokines. Such protective effects of PWS are likely to be, at least in part, mediated via restoration of balance in gut microbial communities.Our study revealed that PWS treatment can ameliorate DSS +


To investigate the safety and therapeutic effect of a modified posterior scleral reinforcement (PSR) in treating high myopia. A total of 85 highly myopic eyes in 47 children  who underwent this modified PSR were included in this study. Axial length, refractive error, best-corrected visual acuity (BCVA), macular scans, and adverse events were recorded before the operation (as a baseline) and in postoperative reviews taken yearly for 5 years.P<0.05) over the 5-year period in all of the children: 6-month (P=0.003), 1-year (P=0), 2-year (P=0), 3-year (P=0), 4-year (P=0), and 5-year (P=0). The axial length was extended. No significant difference was found in refractive error between measurements taken at baseline and at the 5-year postoperative visit in all of the children: 6-month (P=0.51), 1-year (P=0.50), 2-year (P=0.46), 3-year (P=0.56), 4-year (P=0.30), and 5-year (P=0.16). There were significant differences in BCVA between measurements taken at baseline and at the postoperative 5-year visit in all the children: 6-month (P=0), 1-year (P=0), 2-year (P=0), 3-year (P=0), 4-year (P=0), and 5-year (P=0). BCVA improved in 71 eyes (83.52%), remained stable in 14 eyes (16.47%), and declined in none of the children. No serious adverse events occurred before the operation and during the 5-year follow-up period. This was a 5-year research: 50% of the children  participated in the 6-month review, 41% of the children  participated in the 1-year review, 26% of the children  participated in the 2-year review, 16% of the children  participated in the 3-year review, 13% of the children  participated in the 4-year review, and 8% of the children  participated in the 5-year review. Compared with the baseline, axial elongation was significantly changed ( This modified PSR could be a therapeutic treatment for high myopia. Myopia has become a major public health concern, and there is evidence showing a shocking increase in its prevalence . AccordiA large number of treatments have been applied to halt myopia progression in children with low or medium myopia. While these include pharmacological intervention and contact lenses, these therapies achieve little progress in high myopia because axial growth progresses so quickly in these children . PosteriThis study is a retrospective analysis of 47 children   with higThe inclusion criteria applied in this study included high myopia, which was defined as a myopic refractive error ≥ 5.0 diopters (D) and an axial length ≥ 24 mm in subjects aged<8 years with a best-corrected visual acuity (BCVA) inferior to that observed in age-matched children. Children were excluded from the study if they had other ocular diseases that could affect visual function , had systemic disorders that could interfere with the results, or had undergone another ocular surgery, such as refractive surgery, scleral buckle procedure, PSR, or vitrectomy.All operational procedures were conducted by the same surgeon (Dr. Wang). This modified PSR was performed under general anesthesia. At the very beginning of the surgery, a round bivalve scleral buckle (obtained from a donor sclera) with a diameter of 10-12 mm was made by cutting a round scleral buckle out along the direction of the diameter while maintaining the root at 1-1.5 mm . Before First, a temporal bulbar conjunctival incision was made approximately 2 mm from the lower corneal limbus. Second, the inferior oblique and lateral rectus muscles were identified, isolated, and exposed. The two muscles were maneuvered by strabismus hooks while the eyeball was pulled towards the superior nasal side so that the lower temporal part of the sclera could be exposed. Third, a traction suture was placed with 8-0 black silk at the root of the round bivalve scleral buckle. After fixing the buckle's root to the muscle insertion point at the anterior end of the inferior oblique, half of the buckle was placed underneath the inferior oblique , while the other half was placed beneath the lateral rectus . This procedure was accomplished by flattening the scleral buckle with the help of strabismus hooks. Finally, the location of the scleral buckle was checked. Its position relative to the buckle and optic nerve should, in particular, be tested with a strabismus hook. The distance was kept at approximately 3 mm to ensure that each part of the half-round scleral buckle covered the posterior staphyloma without compressing the optic nerve . The conAll children underwent comprehensive ophthalmologic examinations, which included visual acuity, intraocular pressure, axial length, refractive error, best-corrected visual acuity, slit-lamp examination, and fundus examinations at baseline and at every postoperative follow-up visit  .Axial length was measured with an IOL Master  . The statistical outcomes were measured five times, and the mean values were recorded.Refractive error was measured with an autorefractor  and a scientific subjective refractor . Subjective cycloplegic refraction was assessed by experienced optometrists approximately 30 min after 1 drop of tropicamide 0.5% was administered 3 times at 5 min intervals. These data are presented as the spherical equivalent (SE). The statistical outcomes were measured five times, and the mean values were recorded.BCVA was measured with the Snellen acuity test “E,” and the results were converted to LogMAR for statistical analysis.An extraocular muscle examination was performed by the surgery doctors and included eye movements and strabismus checks. The ocular anterior segment was assessed with a slit-lamp, and postoperative adverse events, such as conjunctival injection, edema, and inflammatory response, were recorded. The fundus was examined after the pupil was dilated, and macular scans were observed by indirect ophthalmoscopy (Zeiss). Intraocular pressure (IOP) was measured by a noncontact tonometer (Canon TXF). P<0.05 was considered statistically significant.Postoperative axial length, refractive error, and BCVA were recorded and compared with preoperative measurements using a mixed model with repeated measures at each follow-up visit. The factors that might be associated with myopic progression, such as gender and age, were also taken into consideration and analyzed using the same statistical method. Numerical data are expressed as medians at each time point. All statistical analyses were performed with Stata 10.0 software , andA total of 47 children  were recruited for our study, and their ages ranged from 3 to 15 years . In total, 50% of the children  participated in the 6-month review, 41% of the children  participated in the 1-year review, 26% of the children  participated in the 2-year review, 16% of the children  participated in the 3-year review, 13% of the children  participated in the 4-year review, and 8% of the children  participated in the 5-year review. The observed trends in axial length, refractive error, and BCVA are shown in P<0.05), in detail 6-month (P=0.003), 1-year (P=0), 2-year (P=0), 3-year (P=0), 4-year (P=0), and 5-year (P=0). The average value for axial elongation was 1.32 mm over 5 years, or approximately 0.26 mm per year. According to the mixed model tests, axial length elongation was correlated with baseline age in children who underwent PSR surgery (P<0.05). With regard to gender, a statistical analysis found that there was no significant difference (P=0.09), indicating that gender was not related to axial length.There was a statistically significant difference in axial length between preoperative and postoperative measurements (P<0.05), in detail 6-month (P=0.51), 1-year (P=0.50), 2-year (P=0.46), 3-year (P=0.56), 4-year (P=0.30), and 5-year (P=0.16). According to the mixed model tests, changes in refractive error were associated with gender (P<0.05) but not baseline age (P=0.34). This test revealed that, in boys, refractive error was relatively steady in the following years, whereas, in girls, it increased with age.Comparisons between preoperative and postoperative measurements showed that there was no significant difference in refractive error at the follow-up visit performed after the surgery (P<0.05), in detail 6-month (P=0), 1-year (P=0), 2-year (P=0), 3-year (P=0), 4-year (P=0), and 5-year (P=0). BCVA improved in 71 eyes (83.52%) and remained stable in 14 eyes (16.47%), and none of the records indicated a visual decline in any postoperative patient. BCVA was markedly better at the final follow-up than before the operation. Mixed model tests found that there was an association between BCVA and baseline age in children who underwent PSR surgery (P<0.05), with younger children achieving a better prognosis. There was no statically significant difference in BCVA and gender according to the same analysis (P=0.94).At the follow-up performed after the surgery, we found that there were significant differences in BCVA between preoperative and postoperative measurements (No eye movement restriction or strabismus was found in postoperative extraocular muscle examinations. Conjunctival congestion and edema were observed in all of the children who underwent scleral reinforcement at an early stage, and both conditions were alleviated after several weeks. Postoperative infection was not found in a slit-lamp check in any of the children. No postoperative pathological signs were found in any eye during the fundus examination performed at the last visit. There were no cases of diplopia, hemorrhage, or IOP elevation. None of the eyes lost visual acuity because of the surgery or during the follow-up period.High myopia is characterized by constant axial elongation and gradual thinning of the posterior sclera, both of which are likely due to the weakening of the biomechanical properties of the sclera , 22. ThiThe most critical component of the PSR procedure is that the reinforcement band must precisely surround the posterior pole and be anchored in place to the sclera. If these steps are not successfully performed, the expected effect could be reduced or unfavorable results achieved. Additionally, the magnitude of the strength produced by the enforcement band depends on the material used. Previous studies have demonstrated that a homologous human scleral strip is the best choice for the PSR procedure –36. The In our current study, the results show that the average axial elongation was 0.26 mm per year over the 5-year follow-up period. This change is equivalent to an increase in refractive error of -0.78 D according to calculations performed using a Gullstrand 1 schematic eye. Gerinec et al. reported a mean increase in axial length of 0.2 mm per year after PSR during a follow-up of 3 to 5 years . Minjie The results showed that there was no significant difference in refractive error between measurements taken before and after this modified PSR was performed. This was due to the contribution of the scleral buckle, which slowed down axial length elongation, preventing it from pathologically increasing. A regression analysis indicated that refractive error was not correlated with the distribution of follow-up years after the surgery but was associated with gender. In Zhang's report, the prevalence of myopia was relatively high in girls, and this difference might be associated with differences in outdoor activities between boys and girls . In our In our study, no severe adverse events were observed after the PSR. Normal postoperative responses, such as conjunctival congestion and edema, were present in previous studies of the Snyder-Thompson PSR , 5, 22. Despite the favorable outcomes observed for PSR in the present study, several limitations should be mentioned. These limitations included the retrospective design of the study, the lack of a control group, loss to follow-up, and the relatively small number of enrolled children. All statistical analyses were performed for the above-described defects, and our data confirm that this operation is effective. Future studies should include a control group, a larger sample population, and closer follow-up or should be performed with a prospective design to more precisely compare the effects of these surgical methods in the future.In conclusion, our findings suggest that this modified PSR has the potential to arrest axial elongation in eyes with high myopia. It is a safe operation that requires fewer operational skills and causes little trauma. Further studies are needed to evaluate the long-term safety and effectiveness of this surgery.


Spodoptera litura, is an important pest of crop and vegetable plants worldwide, and its resistance to insecticides have quickly developed. However, the resistance mechanisms of this pest are still unclear. In this study, the change in mRNA and miRNA profiles in the susceptible, indoxacarb-resistant and field indoxacarb-resistant strains of S. litura were characterized. Nine hundred and ten co-up-regulated and 737 co-down-regulated genes were identified in the resistant strains. Further analysis showed that 126 co-differentially expressed genes (co-DEGs)  may play important roles in indoxacarb resistance in S. litura. In addition, a total of 91 known and 52 novel miRNAs were identified, and 10 miRNAs were co-differentially expressed in the resistant strains of S. litura. Furthermore, 10 co-differentially expressed miRNAs (co-DEmiRNAs) had predicted co-DEGs according to the expected miRNA-mRNA negative regulation pattern and 37 indoxacarb resistance-related co-DEGs were predicted to be the target genes. These results not only broadened our understanding of molecular mechanisms of insecticide resistance by revealing complicated profiles, but also provide important clues for further study on the mechanisms of miRNAs involved in indoxacarb resistance in S. litura.The tobacco cutworm,  Spodoptera litura (Fabricius), is an important polyphagous insect pest that causes widespread economic damage to vegetables and other crops, such as soybean, tomato, sweet potato, groundnut and cotton2. Over many years, the control of S. litura has traditionally relied on sprays of various insecticides3. Many field populations of S. litura have developed high resistance to multiple insecticides, including organophosphate, carbamate, pyrethroids and some newer chemistry insecticides such as indoxacarb, abamectin, emamectin benzoate and chlorantraniliprole6. The effort to control this pest is becoming exceedingly challenging all over the world. Therefore, understanding the mode of action and resistance mechanisms of insecticide are extremely important for us to design effective resistance management strategies. Also, identifying the molecular mechanism responsible for insecticide resistance will provide new opportunities for the development of novel strategies for insect pest control.The tobacco cutworm, 7. Metabolic resistance has been reported worldwide and mostly associated with an increased level of detoxification enzymes (such as cytochrome P450 (P450), carboxy/cholinesterase (CCE), and glutathione S-transferase (GST)) in insecticide-resistant populations8. Functional genomics tools have been used to diagnose the genetic basis of pesticide resistance in arthropods9. Such studies showed that insecticide resistance is more complex than thought before, mediated by multigenic systems that involve large parts of the insect genomes. However, due to the large number of S. litura genes encoding detoxification enzymes, pinpointing those responsible for resistance remains challenging10.Studying insecticide resistance makes it possible to classify pest adaptations into two main mechanisms: degradation of metabolic detoxification with elevating enzyme activities or insecticide target insensitivity due to point mutations12. As a key component in post-transcriptional gene expression regulation, miRNAs play important roles in many physiological processes, such as metabolism, growth, development, behavior and apoptosis13. Some research has shown that miRNAs were involved in the formation of insecticide resistance in arthropods. For example, it has been demonstrated that miR-71 and miR-278-3p could regulate the pyrethroid resistance in Culex pipiens14 and Culex pipiens pallens15, respectively. MiR-276 and miR-3016 were found to be responsible for spirotetramat resistance in Aphis gossypii16, Tci-miR-1-3p was involved in cyflumetofen resistance in Tetranychus cinnabarinus17. However, in S. litura, there is still insufficient information regarding the relationship between miRNAs and insecticide resistance.MicroRNAs (miRNAs) are endogenous small (about 22 nucleotides) non-coding RNAs that regulate gene expression through binding to the 3′untranslated region (3′UTR) of target mRNA, and causing inhibition of translation or mRNA degradation at post-transcriptional levelS. litura. Then we analyzed the expression profiles of the mRNAs and miRNAs from three strains and predicted the targets of miRNAs. Co-differentially expressed genes (co-DEGs) and co-differentially expressed miRNAs (co-DEmiRNAs) in the two resistant strains were screened, and the expression profiles were then validated by quantitative real-time PCR (qPCR) techniques. We further focused on co-DEGs related to insecticide resistance. Finally, the regulatory relationships between co-DEmiRNAs and insecticide resistance-related co-DEGs were analyzed. The aim of the present study, therefore, was to clarify the indoxacarb resistance mechanisms and the regulatory mechanisms of miRNAs in S. litura.In this study, we employed the high-throughput sequencing platform-Illumina HiSeq 2500 to complete the mRNA and small RNA (sRNA) sequencing from the fourth-instar larva of susceptible strain (SS), lab indoxacarb-resistant strain (InRS) and field indoxacarb-resistant strain (FInRS) of 50 of SS, InRS and FInRS were 0.64 mg/L, 37.37 mg/L and 25.65 mg/L, respectively. The InRS and FInRS exhibited 58.39-fold and 40.08-fold resistance against indoxacarb compared to the SS, respectively | ≥ 1 and P < 0.05 as the cut-off between resistant and susceptible strains. Comparing with the SS, 1576 up- and 1227 down-regulated, 2606 up- and 1781 down-regulated genes were identified in the InRS and FInRS of S. litura, respectively  Fig.  and 9 ofSlituP450-003) to 66.46-fold (SlituP450-018) and 2.78-fold (SlituP450-037) to 199.98-fold (SlituP450-085) in the InRS and FInRS, respectively  to 37.20-fold (SlituCOE073) and 2.72-fold (SlituCOE111) to 10.01-fold (SlituCOE073) in the InRS and FInRS, respectively  (Table SlituGST20 and SlituGST38) were 4.24-, 72.65-fold and 29.47-, 17.30-fold in the InRS and FInRS, respectively, all belonging to class epsilon.  in the InRS and FInRS Table . The folThere are several other insecticide resistance-related genes that were up-regulated in the InRS and FInRS, such as 10 UDP-glucuronosyl transferase (UGT), 9 ATP-binding cassette (ABC) transporter and 1 aminopeptidase N (APN) genes. These genes were detected to be co-up-regulated in the InRS and FInRS , Table 18. Indoxacarb can be metabolized by insect esterases or amidases to a N-decarbomethoxylated metabolite (DCJW), which is a more active sodium channel blocker than indoxacarb, resulting in paralysis and death of target pest species20. Because of its safety to mammals and non-target organisms, favorable environmental and residue properties, broad spectrum and rapid inhibition of insect feeding making indoxacarb a powerful new insecticide for crop protection18. However, due to intensive use of indoxacarb, many studies have shown that several insects have developed resistance to indoxacarb in recent years, including Choristoneura rosaceana21, Musca domestica22, Plutella xylostella24, Spodoptera exigua25 and Helicoverpa armigera26. In S. litura, field populations of S. litura collected from various areas of Pakistani during 2004–2006 and 2009–2011 developed indoxacarb resistance from 3- to 95-fold3 and 7- to 87-fold27, respectively. Monitoring insecticide resistance of S. litura showed that the field populations of Hunan Province had produced 1.92- to 53.4-fold resistance to indoxacarb during 2010–2012 and 2014–201628, and the field populations of Sichuan Province had developed 2.2- to 31.0-fold resistance to indoxacarb during 2014–201629.Indoxacarb is a novel oxadiazine insecticide which has good field activity against a number of lepidopteran pests, as well as certain homopteran and coleopteran pestset al.29 identified two point mutations (F1845Y and V1848I) in P. xylostella, and Gao et al.25 identified one point mutation (L1014F) in S. exigua, which have been proven to confer high levels of resistance to indoxacarb. Even though enhanced target site insensitivity is important for the resistance of insects, detoxification enzymes are also important factors for the metabolism of insecticides. Shono et al.22 showed that P450 was involved in the resistance to indoxacarb in M. domestica. Sayyed and Wright30, and Nehare et al.31 found that esterase and GST were related to resistance to indoxacarb in P. xylostella. Gao et al.25 also suggested that carboxylesterase and GST were major factors leading to indoxacarb resistance in S. exigua. What is more, it was demonstrated that the increased activities of carboxylesterase and P450 were important in conferring indoxacarb resistance in S. litura28. However, the indoxacarb resistance mechanism of S. litura remains unclear at molecular level.The mechanisms mediating resistance to indoxacarb have been studied in several insects. Some mutations in the sodium channel gene have been shown to confer target site insensitivity to the neurotoxic effects of indoxacarb. Wang 32. In the previous research, Cheng et al.10 have shown that 4 P450 , 2 CCE (SlituCOE057 and SlituCOE058) and 2 GST (SlituGST07 and SlituGST20) genes are linked to tolerance of imidacloprid through induction by imidacloprid and knockdown experiments in S. litura. In the current study, 19 P450 genes were up-regulated in the indoxacarb-resistant strains and widely distributed in clan 3 , clan 4  and clan M . It has been shown that the members of the clan 2, clan 3, clan 4 and clan M of P450s are commonly connected with environmental response/detoxifying functions against xenobiotics and phytotoxins in most arthropod species35. Seven CCE genes, including 3 lepidopteran esterases , 1 α-esterase (SlituCOE009) and 3 integument esterases , and 2 epsilon GST (SlituGST20 and SlituGST38) genes were also up-regulated in the indoxacarb-resistant strains. In insects, the lepidopteran and α classes CCE, and epsilon classe GST have been shown to be associated with xenobiotic detoxification36. These results confirmed our prediction that overexpressed P450, CCE and GST genes were responsible for indoxacarb resistance in S. litura. It’s worth noting that only SlituGST20 may be involved in both imidacloprid and indoxacarb resistance, indicating that different types of insecticide resistance is mediated by different detoxification genes in S. litura.Among the elucidated metabolic mechanisms of resistance, the most common ones include enhanced detoxification of insecticide by up-regulated P450, CCE and GST37. Overexpressed P. xylostella UGT2B1738 and T. cinnabarinus UGT201D339 have been shown to be involved in insecticide resistance. In addition to enzymes related to metabolism and conjugation, a number of transporter families, of which ABC transporters are the best studied, also play an important role in xenobiotic tolerance40. Sun et al.41 suggested that ABC transporters might be involved in resistance to multiple insecticides in Laodelphax striatellus. What is more, APN has been shown to function as Cry protein receptor in insects42 and might be involved in the response to different classes of xenobiotics in S. litura10. In this case, the over-expression of 10 UGT, 9 ABC transporter and 1 APN genes may also associate with the detoxification process of indoxacarb in S. litura.In addition to the well-known detoxification gene families involved in insecticide resistance, we also revealed that other insecticide-related genes had significant higher expression levels in the resistant strains. UGT as biotransformation enzymes, widely distributed within living organisms and viruses, were presumed to originally participate in the detoxification processS. litura to better tolerate insecticides might also be the consequence of the induction of other proteins involved in a wide range of functions. The major facilitator superfamily (MFS), belonging to secondary active membrane transporters, can transport a wide range of small solutes  in response to chemiosmotic ion gradients in humans. The roles of MFS on adapting stress from host plant shift and acaricide exposure were reported in Tetranychus urticae43. Sialin is a member of the MFS of transporters44. In the present study, up-regulation of sialin gene in S. litura may result in a higher efflux of insecticides out of S. litura cells and developing of insecticide resistance. Serine protease, secreted by pancreas in mammals, mainly function in two aspects: protein digest and hydrolysis as well as activation of all proenzymes secreted by pancreas45, which is important in the cellular and the humoral arm of invertebrate immune response. It had been reported that up-regulated SP genes were related to deltamethrin resistance in C. pipiens pallens46. Intracellular proteases might play a role in protein biosynthesis or modified conformation of enzymes as part of this induction process47. This mechanism may involve increased supply of precursor amino acids from proteolytic degradation products to the intracellular pool, prior to synthesis of detoxifying enzymes in S. litura following insecticide exposure. This hypothesis could be supported by another fact that 6 up-regulated SP genes were found in S. litura responded to indoxacarb in our study. Many studies on cuticle protein suggest that insecticide resistance could manifest as a slower rate of penetration due to higher protein and lipid content in the cuticle and/or greater sclerotization and was caused by increased cuticle genes expression48. The overexpression of so many CP genes in this study indicated that this gene family may be involved in indoxacarb resistance in S. litura.By revealing that several other genes with a broad range of biological functions are similarly affected by insecticides, our results suggest that the ability of S. litura since the other 55.3% of total co-DEGs were up-regulated. It is generally recognized that overexpressed genes may play more important roles than down-regulated genes in the insecticide resistance. The decreased expression of some metabolic detoxification genes might result from the responses to various endogenous and exogenous compounds, or path of physiological signals49. Many studies have deduced the balance mechanism of up- and down-regulated genes, including an adaptive homeostatic response that protects the cell from the deleterious effects of oxidizing species, such as nitric oxide and arachidonic acid metabolites from catalytic and/or metabolic enzymes50. In return, this also could be a pathological response to inflammatory processes and a need for the tissue to utilize its transcriptional machinery and energy for the synthesis of other components involved in insecticide resistance51.A total of 737 genes were down-regulated  in the resistant strains. This phenomenon is very understandable since all living organisms have limited energy inputs. Reduced expressions in these genes would save some energy for resistant 52. In M. domestica, 1316 genes were identified as being up-regulated in the multiple insecticide resistant strain in comparison to the susceptible strains by transcriptome analysis, and the majority of these up-regulated genes fell within the structural classification of proteins (SCOP) categories of metabolism, regulation and intracellular processes53. A similar whole transcriptome study has been carried out in Culex quinquefasciatus. The results showed that 367 genes were found to be up-regulated in the highly permethrin-resistant strain, and all P450 genes were up-regulated by at least twice54. Bai et al.55 also revealed that P450 genes (CYP9) were highly expressed in pesticide-exposed Cimex lectularius populations by transcriptome and qPCR analysis. In this study, 910 co-up-regulated and 737 co-down-regulated genes were identified in the indoxacarb-resistant strains through mRNA sequencing, and the roles of 126 detoxification-associated genes (107 co-up-regulated and 19 co-down-regulated) in indoxacarb resistance were systematically analyzed. These results provide clues to the identification of potential detoxification genes involved in indoxacarb resistance in S. litura. Although the two resistant strains have different genetic background, it can be more reasonably screened to obtain insecticide resistance-related genes that reflect the real situation in the field. A similar approach has been used to study the molecular mechanisms of spirodiclofen resistance in T. urticae56.Analysis of mRNA through high throughput expression profiling using transcriptome analysis methods has provided considerable advances in understanding the molecular base of resistance in insects57. In C. pipiens, 100 known miRNAs and 42 novel miRNAs were identified, and 28 miRNAs were differentially expressed in the susceptible and deltamethrin-resistant strains14. Seventy-five known miRNAs and 64 novel miRNAs were also identified in the susceptible and fenpropathrin-resistant strains of T. cinnabarinus, including 12 differentially expressed miRNAs17. In this study, we identified 91 known miRNAs and 52 novel miRNAs in S. litura, 10 of which were co-differentially expressed in the InRS and FInRS. This data has detected significantly more miRNAs than the early report by Rao et al.13, in which only 58 miRNAs were identified among different developmental stages in S. litura. The reasons that we can detect increased numbers of miRNAs in our study compared to Rao et al.13 were the availability of S. litura whole genome, the improved methods and the upgraded miRBase database.To date, more than 30,000 miRNAs have been found in over 100 organismsS. litura. Among 126 indoxacarb resistance-related co-DEGs, 33 up-regulated co-DEGs were predicted target genes of 5 down-regulated co-DEmiRNAs, 4 down-regulated co-DEGs were predicted target genes of 5 up-regulated co-DEmiRNAs. With the deepening of the study of miRNA function, some miRNAs have been shown to be associated with insecticide resistance by regulating resistance related genes in insects and mites. Hong et al.14 and Lei et al.15 indicated that miR-71 and miR-278-3p were involved in pyrethroid resistance by targeting CYP325BG3 and CYP6AG11 in C. pipiens and C. pipiens pallens, respectively. Zhang et al.17 demonstrated that Tci-miR-1-3p could regulate the cyflumetofen resistance through TCGSTM4 in T. cinnabarinus. Ma et al.58 also indicated that miR-92a regulates pyrethroid resistance through its interaction with a cuticular protein gene, CpCPR4 in C. pipiens pallens. Thus, our results indicated that co-DEmiRNAs of S. litura may function in the formation of the indoxacarb resistance through regulating the insecticide resistance-related co-DEGs.In general, miRNAs play important gene-regulatory roles by targeting the mRNAs of protein coding genes and repressing their post-transcriptional properties. In this way, down-regulation of a miRNA indicates increased activity of its target gene. It is an important step to identify the target genes of miRNAs for understanding their roles in gene regulatory networks. Therefore, we analyzed the relationship between indoxacarb resistance-related co-DEGs and co-DEmiRNAs in S. litura. A total of 1647 co-DEGs (910 co-up-regulated and 737 co-down-regulated) and 143 miRNAs, including 10 co-DEmiRNAs (5 co-up-regulated and 5 co-down-regulated) were identified in the resistant strains of S. litura. Our data revealed that indoxacarb selection strongly affected the transcription levels of 126 detoxification-associated genes  involved in indoxacarb resistance. Further analysis showed that 37 indoxacarb resistance-related co-DEGs were predicted to be the target genes of 10 co-DEmiRNAs, and indicated that these miRNAs may regulate the indoxacarb resistance through these indoxacarb resistance-related genes in S. litura. Overall, the present study help us better understand the indoxacarb resistance mechanisms and the regulatory mechanisms of miRNAs in S. litura at the molecular level. Functional analysis of these resistance related genes and verification of the regulatory relationships between these miRNAs and their target genes are needed to further elucidate the indoxacarb resistance mechanisms in S. litura.In conclusion, mRNA and sRNA libraries were constructed and sequenced in the SS, InRS and FInRS of S. litura was obtained from the Institute of Zoology, Beijing, China. The field population of S. litura was collected from the field crops of Changsha City, Hunan Province, China. There was no specific permission required for these collection activities because this insect is a kind of agriculture-harmful pest and distributes worldwide. We confirm that the field collection did not involve endangered or protected species.The laboratory population of S. litura was obtained from the Institute of Zoology, Beijing, China and kept in the laboratory under artificial diet and climate without any insecticide exposure. InRS: the laboratory resistant strain was selected with indoxacarb in the laboratory from the SS. FInRS: the field indoxacarb resistant strain was collected from the field crops of Changsha City, Hunan Province, China. The SS/InRS and FInRS have different genetic background. The rearing conditions were set as: 25 ± 2 °C, 65 ± 5% relative humidity (RH) and a 14 h light (L): 10 h dark (D) photoperiod.SS: the laboratory population of S. litura using the artificial diet dipping method59. Briefly, indoxacarb  was dissolved in sterilized water to at least 5 concentrations and the mortality was kept at 20–80%. The artificial diet was cut into an area of 2 cm2 and a thickness of 5 mm, and dipped into the indoxacarb solution for 10 s, including sterilized water as control. These artificial diets were air dried at room temperature for 5–10 min. Then, fifteen larvae were placed on each treated artificial diet. Each dose was performed in three replicates. The bioassays were kept at 25 ± 2 °C, 65 ± 5% RH and 14:10(L:D) photoperiod. Mortality was recorded 24 h after exposure for indoxacarb. Larvae were considered dead if they failed to make a coordinated movement when prodded with a brush. LC50 values were calculated via probit analysis using PoloPlus software .Bioassays were conducted with fourth-instar larvae of Total RNAs were extracted from fourth-instar larvae of SS, InRS and FInRS using TRIzol reagent , respectively. RNA degradation and contamination was monitored on 1% agarose gels. RNA purity, concentration and integrity were measured using the NanoPhotometer spectrophotometer , Qubit RNA Assay Kit in Qubit 2.0 Flurometer  and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system , respectively.S. litura10 using Hisat2 v2.0.560.Qualified total RNAs from three strains  were used for preparing sequencing libraries using NEBNext Ultra RNA Library Prep Kit for Illumina  following manufacturer’s recommendations. The library preparations were sequenced on an Illumina Hiseq 2500 platform and 125 bp paired-end reads were generated at the Novogene Bioinformatics Institute  after the clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq SR Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. In order to obtain clean reads, the low-quality reads, adaptor sequence, ploy A or T or G or C and duplication sequence were removed from raw data. Then, clean reads were aligned to the reference genome of 61. DEGs analysis was performed using the DESeq2 R package (1.16.1)62. Genes with an adjusted P < 0.05 and |log2(fold change)| ≥ 1 were assigned as differentially expressed. Gene Ontology (GO) database63 was used to identify functional modules of DEGs (P < 0.05).Gene expression levels were estimated by FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced)S. litura using Bowtie software64. Then, the matched sRNAs were compared with the mature miRNAs in miRBase 20.0 (http://www.mirbase.org/) to looking for known miRNA of S. litura, only perfectly matches were accepted and counted. Next, the remained sRNAs were compared with Repeatmasker and Rfam database (ftp://selab.janelia.org/pub/Rfam) to remove protein-coding genes, repeat sequences, ribosomal RNA (rRNA), small cytoplasmic RNA (scRNA), transfer RNA (tRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA). At last, the remained sRNAs were used to predict the novel miRNA through the two available software miREvo65 and mirdeep266.Qualified total RNAs from three strains  were used as input materials for the sRNA libraries. Sequencing libraries were generated using NEBNext Multiplex Small RNA Library Prep Set for Illumina  following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, total RNAs were ligated to 5′ and 3′ adaptors, then first strand cDNA was synthesized by reverse transcriptase. After PCR amplification of the cDNA, the amplified PCR products within 140–160 bp were separated and purified by a 8% polyacrylamide gel. Libraries quality were assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 2500 platform and 50 bp single-end reads were generated at the Novogene Bioinformatics Institute . Clean reads were obtained by removing reads containing ‘N’ (an unrecognized base) at 10% or higher, with 5′ adapter contaminants, without 3′ adapter or the insert tag, containing ploy A or T or G or C and low-quality reads. After removing reads with a sequence shorter than 18 nt or more than 35 nt, clean reads were mapped to the reference genome of 62. Corrected P < 0.05 was set as the threshold for significantly differential expression. The miRNAs expression levels were estimated by TPM (transcript per million)67. Predicting the target genes of miRNAs were performed by miRanda68 and PITA69. In addition, GO analysis was also used to explore the functions of the predicted target genes (P < 0.05)63.DEmiRNAs were performed using the DESeq2http://frodo.wi.mit.edu/) and Primer Premier 5.0 . Arginine kinase (AK) and U6 small nuclear RNA (U6) were used as stable reference genes for qRT-PCR assays of mRNA and miRNA, respectively70. The qPCR reactions were performed on Bio-rad Detection iQ2 System  with 20 μL reaction mixtures containing1 μL of diluted cDNA, 10 μL TransStart Tip Green qPCR SuperMix , and 1 μL of each primer (10 μM). The optimized qPCR protocol used for the amplification was: 94°C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. Melting curve analysis was performed from 60 to 95 °C to ensure consistency and specificity of the amplified product. The qPCR experiments were conducted according to minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines71. The expression levels of mRNA and miRNA were calculated using the 2−ΔΔCt method72. Three biological and two technical replicates were performed for each mRNA and miRNA. The qPCR results were analyzed by an independent-sample t-test with a significance level of P < 0.05 using SPSS 23.0 software .To verify the reliability of RNA sequencing, twenty DEGs and all 10 DEmiRNAs were selected to validate their expression pattern by qPCR. Total RNAs were reverse transcribed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit . Primers of mRMA (Supplementary Table Supplementary InformationSupplementary Table S5Supplementary Table S7Supplementary Table S8


Medial meniscal extrusion (MME) is an important marker of knee osteoarthritis (KOA) progression. The purposes of this study were: 1) to determine whether there are morphological differences between CT- and MRI-derived tibial plateau models; and 2) to determine whether measurement of MME volume and width using an MRI-derived tibial model is as accurate as measurements on a CT-derived tibial model.This was a cross-sectional study that enrolled ten participants with medial KOA (Kellgren-Lawrence grade 1 to 3). Primary outcome was surface difference of the medial tibial plateau between CT- and MRI-derived models. Furthermore, volume and cross-sectional area of the medial tibial plateau were compared between CT- and MRI-derived models. Measurements of MME volume and width were compared between CT- and MRI-derived tibial models.3 and 916.2  mm3, respectively (p = 0.938). The MME widths measured on CT- and MRI-derived models were 4.2  mm and 4.5  mm, respectively (p = 0.967).Minimal and maximal surface differences of the medial tibial plateau between the CT- and MRI-derived models were − 0.15  mm  and 0.24  mm, respectively. There were no significant differences in volume and cross-sectional area of the medial tibial plateau between CT- and MRI-derived tibial models. The MME volumes measured on CT- and MRI-derived models were 942.6  mmCT- and MRI-derived models of the medial tibial plateau did not show significant morphological differences. Both CT- and MRI-derived tibia can be used as a reference to measure MME in early-to-moderate medial KOA. Medial meniscal extrusion (MME), with or without meniscal injury, is an important marker of knee osteoarthritis (KOA) progression. Both Kawaguchi et al.  and YanaOur ability to assess the MME and osteophytes around the tibial plateau three-dimensionally may be limited. To improve longitudinal assessments of changes in MME volume and width, effects of the osteophytes should be eliminated. Therefore, Computed tomography (CT) could represent a valid alternative since CT demonstrates sharp bone contours and allows automatic or semi-automatic segmentation in a reproducible manner , 6. In aIn fact, the contour of the tibial plateau serves as a reference point for measuring MME. Several studies chose the outermost margin of the tibial plateau as the reference after researchers manually excluded any osteophytes , 12, 13.This study aimed to determine: 1) whether there are morphological differences between CT- and MRI-derived tibial plateau models, 2) whether measuring MME volume and width on MRI-derived tibial models is as accurate as measurements using CT-derived tibial models. The hypotheses of this study were: 1) volume and area of the CT-derived tibial plateau would be larger than those derived from MRI; and 2) MME width measured on 2D MRI slices is inaccurate compared with 3D measurement on CT-derived tibial models combined with MRI-derived meniscal models.This was a cross-sectional study based on image data obtained as baseline data in a randomized controlled trial (RCT) investigating the effect of exercise therapy in patients with KOA. This RCT was approved by the institutional review board of Hiroshima Prefectural Rehabilitation Center and Hiroshima International University . Participants of this study were recruited from patients consulting the Department of Orthopaedics at Hiroshima Prefectural Rehabilitation Center. Informed consent was obtained from each of the individual participant included in this study in accordance with the Helsinki Declaration.Inclusion criteria were Japanese aged from fifty to eighty years old, primary KOA, and KL grade from 1 to 3. Exclusion criteria were valgus KOA, secondary KOA, a history of knee surgery or knee injury, a history of other somatic diseases that could pose a risk, mental disorders, and communication difficulty. 327 patients were assessed for eligibility and 39 patients met the criteria. Ten subjects in this study were selected based on the additional procedure to choose three or four patients randomly from the stratified pools of patients based on the KL grade 1–3.CT images were obtained using a clinical X-ray CT scanner . CT scan was taken using axial slices, kilovoltage: 120 kVp, tube current: 70 mA, exposure time: 500 ms, exposure: 70 mAs, slice thickness: 0.50 mm, sampling 150 × 150 mm in-plane sampling, and imaging matrix: 512 × 512. MRI images were obtained using a 1.5 Tesla MRI , and knee coil . MRI were obtained as coronal slices of proton density sequences, slice thickness: 2.0 mm, intersection gap: 0 mm, slice resolution: 180 × 180 mm, imaging matrix: 384 × 384, TE time: 11 ms, TR time: 3810 ms. Participants were in the supine position with the foot elevated so that the examined knee was fully extended.Manual segmentation was utilized to differentiate osteophytes and the other tissues using 3D modeling software . Geometric bone models and medial meniscus (MM) models were created by segmenting the exterior cortical bone edges and MM edges. Created 3D models were converted to polygonal surface models. Smoothing was applied using a reverse engineering software .A tibial coordinate system was embedded on the CT-derived tibia, which then was best-fitted to the MRI-derived tibia in order to share a common local coordinate system. A single experienced researcher embedded the local tibial coordinate system onto the CT-derived tibial models using commercial software . On the CT-derived model, a virtual rectangle parallel to the tibial plateau plane was fitted onto the tibial plateau contours at the top of the fibular notch of the tibia in order to avoid osteophytes on the osteoarthritic tibial plateau . Four siSurface differences and volume of the medial tibial plateau model were calculated using Geomagic Studio. A model of the medial tibial plateau was created by cutting the original tibial bone model at the level of 10.0 mm inferior to the origin parallel to the ZX plane and at the original XY plane Fig. . The ost2D MME width was measured using image processing software . 2D MME width was defined as the distance from the most extruded edge of the MM to the edge of the medial tibial plateau . The corSample size of this study was calculated using a power analysis program . A power analysis was conducted using the result of the previous study . 0.82 ± All variables were tested with Shapiro-Wilk test in order to determine the normality. Mean and 95% confidence interval were used to assess the demographic characteristics. Two-sample t-test was used to test each variable of the CT- and MRI-derived models. Linear regression analysis was carried out in order to test consistency of the medial tibial plateau volume and the cross-sectional area between the CT-derived models and MRI-derived models. Next, linear regression analysis was carried out to test consistency of the MME volume and width between the CT-derived models and MRI-derived models. In both analyses, data from the CT-derived model were dependent variables and data from the MRI-derived model were independent variables. Lastly, linear regression analysis was performed in order to assess the consistency between the 3D MME width and the 2D MME width obtained from an MRI slice. Statistical analysis was performed using commercial software . The level of significance was set at α = 0.05.2, respectively  of the participants were 69.5   years, 157.8  cm, 61.9  kg, and 25.1  kg/mICC for 2D MME width for intra-researcher agreement was 0.990 . ICC for measuring the MME volume was 0.998 . ICC for measuring the MME width was 0.983 .All the participants had osteophyte in the medial tibial plateau, and none of ten had small osteophytes at 10.0 mm below the tibial plateau plane. All the participants except one could fully extend their knees. One participant with KL grade 3 had flexion angle of 4.5° by calculating with 3D bone models during MRI acquisition. MME volume and MME width using the CT- and MRI-derived tibia, and cross-sectional area of the medial tibial plateau using the CT- and MRI-derived tibia were normally distributed Table . However3 (median (inter-quartile range)) and 12,628 (4373) mm3 , respectively  and 1493  mm3 , respectively. Therefore, there were no significant differences in the volume and cross-sectional area of the medial tibial plateau between the MRI-derived and CT-derived tibial models. Linear regression analysis demonstrated that the volume of the medial tibial plateau of the CT- and MRI-derived models showed a high consistency  = 8015, p < 0.001) with adjusted R2 of 0.999  and 0.23  mm, respectively Table . The vol, 0.16 mm3 and 916.2  mm3, respectively  . MME width measured on a 2D MRI slice was 5.5  mm. In addition, linear regression analysis demonstrated that the MME volumes on the CT- and MRI-derived models showed a high consistency  = 2493, p < 0.001) with adjusted R2 of 0.996  = 51.6, p < 0.001) with adjusted R2 of 0.835.The MME volumes of the CT- and MRI-derived models were 942.6  mm2 was lower than that of the MME width on CT- and MRI-derived models.The most important findings in this study were that surface differences of the medial tibial plateau ranged from − 0.15  to 0.24 , and that there was no significant difference between the MME volume calculated using the CT- and MRI-derived tibial models, and no significant difference between the MME widths calculated using the CT- and MRI-derived tibia. The consistency of the MME volume and the MME was excellent on linear regression analysis. 2D MME width was 5.5  mm, and the coefficient of determination adjusted RAccurate MME measurement requires accurate contours of the tibial plateau with the longitudinal osteophytes taken into consideration. Neubert et al.  showed tLongitudinal comparison of the MME requires reasonable management of osteophyte growth over time. Hayeri et al.  observedThere has not been a previous study that compared MME measurements obtained from CT- and MRI-derived tibial bone models combined with an MRI-derived MM model. In patients with medial KOA, MME width on 2D MRI measured 4.3 ± 2.5 mm . MME widIn this study, there was no significant difference in MME measurements between the CT- and MRI-derived tibial bone models. However, the average surface difference of 0.24  mm at maximum would likely be a source of systematic error and should be taken into consideration when comparing MME measurements between the CT- and MRI-derived tibial bone models. From the above, the MRI-derived tibial models provided reasonably accurate measurements of the MME volume and width after removing the influence of tibial plateau osteophytes. The internal validity of this study was high, and the methods used in this study are available for MME measurement in the early-to-moderate stages of primary medial KOA.This study has a few limitations. First, the MRI sequences of this study were not specialized for analyzing bone morphology. This study employed a clinical sequence for analyzing medial meniscal disorder. Surface differences were similar to the findings of previous studies. Secondly, this study employed coronal slices in the MRI and the slice pitch was 2.0 mm. This may have affected the anterior or posterior margin of the MM, which can cause some error in volume and lesser extent in distance. Therefore, caution is necessary when comparing data from different studies due to errors caused by the segmentation method on MRI and when comparing 2D and 3D measurements of the MME. Thirdly, manual segmentation of the MM was done by single researcher. One observer segmentation has an advantage in reducing inter-observer errors, which is desired in a small study with 10 samples. However, it has a limited generalizability. Fourthly, this study applied the level of 10.0 mm as the reference cut point of the tibial plateau model. All the tibia in this study had no visual osteophyte at the level. However, careful attention would be needed when analyzing the MME with greater osteophyte in the tibial plateau.In conclusion, this study showed that the medial tibial plateau does not demonstrate significant morphological difference between the CT- and MRI-derived models, and that both models can be used as a reference to measure the MME in early-to-moderate medial KOA. This study showed the validity of the MME measurement method using the MRI-derived 3D tibial models after excluding the influence of tibial plateau osteophytes. Further studies are required to determine longitudinal changes in MME after eliminating the effects of osteophyte growth. This study concluded that the morphology of the medial tibial plateau does not demonstrate significant differences between CT- and the MRI-derived models, and that both the CT- and MRI-derived tibial model can be used as a reference when measuring MME in early-to-moderate medial KOA.


Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism  Thioredoxins (Trxs) participate in dithiol/disulfide exchange reactions that effect structural and functional changes in target proteins via reversible change in the redox state of selected cysteine (Cys) residues. Canonical members of the Trx family contain the common amino acid sequence motif WCGPC in the active site with two strictly conserved Cys that target proteins capable of reversibly forming a disulfide bridge. The Cys are reduced to the sulfhydryl form by a Trx reductase (TR) . In manyAeropyrum pernix [Pyrococcus horikoshii [Methanosarcina acetivorans [Sulfolobus solfataricus [Methanocaldococcus jannaschii [Thermoplasma acidophilum [M. jannaschii TR (MjTR). T. acidophilum TR (TaTR), by contrast, is linked to NADH pyridine [The structural dynamics and functional mechanisms of NTRs have been extensively studied in bacteria and eukaryotes. In archaea, functional studies of the Trx system are available for several species, including m pernix , Pyrococrikoshii , Methanotivorans ,7, and Sataricus . The arcnnaschii  and Therdophilum , where tpyridine ,11.Methanosarcina mazei, and (ii) to determine the X-ray crystal structure of its NTR. M. mazei is an anaerobic ecologically important methanoarchaeon with a broad substrate spectrum, able to generate methane using H2 and CO2, methanol, or methylamines as well as acetate as an energy and a carbon source [The present study was conducted (i) to apply in silico analysis for identifying members of the TS in the mesophilic n source ,13. It rM. mazei strain Gö1 (MM2353) was cloned into pET28a, resulting in a cleavable N-terminal 6xHis-tagged fusion protein. Protein was produced in BL21pRIL Escherichia coli cells upon induction with 0.2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) at 22 °C. Cells were disrupted by sonication in 20 mM Tris-HCl buffer, pH 7.6, containing 300 mM NaCl, 10% glycerol, and 1 mM phenylmethylsulfonyl fluoride. The crude extract was clarified by centrifugation at 40,000× g for 1 h. The enzyme was purified using nickel-nitriloacetic acid (Ni-NTA) chromatography  and digested with thrombin  in buffer, 20 mM Tris-HCl pH 8, 150 mM NaCl, and 2 mM CaCl2, at room temperature overnight. The last step of purification involved gel filtration chromatography (Sephacryl S-300) using 10 mM Tris-HCl pH 7.6, 100 mM NaCl, and 2 mM 2-mercaptoethanol buffer. For protein molecular weight estimation by size exclusion chromatography, the column was calibrated with the Gel Filtration Standard mixture from Bio-Rad.The full-length NTR of Thermoplasma acidophilum (Protein Data Bank (PDB) code 3CTY) [One microliter of a 15 mg/mL protein solution in buffer, 10 mM Tris-HCl pH 8 and 50 mM NaCl, was mixed with 1 µL of a solution containing 0.1 M sodium acetate, pH 4.8, and 1 M ammonium chloride. Protein crystals appeared after 4 days of incubation at 25 °C on a sitting drop crystallization plate. Crystals were transferred to a solution containing Paratone Oil before flash freezing. Diffraction data were collected at a wavelength of 1 Å on the X06DA beamline of the Swiss Light Source synchrotron . The diffraction data were processed and scaled using XDS and XSCALE programs . The MmNde 3CTY)  was thende 3CTY) , alternade 3CTY) . Data code 3CTY) . Atomic −1. The X-ray wavelength was set at 1.0 Å, equivalent to 12.4 keV. The scattering data were analyzed using the programs Chromixs [SAXS data were collected at the B21 beamline at the Diamond Light Source using an online SEC-HPLC system coupled SAXS setup. In line SEC-SAXS was performed using an Agilent 1200 HPLC system equipped with a 2.4 mL Superdex 200  column. Forty-five microliters of a protein sample at 5 mg/mL were loaded onto the size exclusion column equilibrated in 20 mM Tris-HCl pH 7.6, 150 mM NaCl, and 2 mM DTT. Data were collected all along the chromatogram at 3 s per frame, and recorded on a Pilatus 2M detector with a fixed camera length of 4.014 m, covering a scattering vector (q) range from 0.0032 to 0.38 ÅChromixs .Homolog protein sequences were retrieved from the National Center for Biotechnology Information’s database using the Blastp program . ProteinM. mazei Gö1 revealed an open-reading frame (Locus tag MM_RS12200) that encoded a protein annotated as NADPH-dependent thioredoxin reductase (MmNTR). The amino acid sequence of the NTR-translated gene contained linear motifs for the binding of FAD (GGGPAG) and NADPH (GGGNSA and HRRDHLK) and a CxxC redox motif (M. acetivorans C2A NTR (MaNTR) showed 83% identity and 92% similarity at the protein sequence level. Two other archaeal TR proteins have been included in the sequence analysis as they are of interest: the TR of M. jannaschii (MjTR) that employs F420 as an electron donor [T. acidophilum (TaTR), which was initially described as a pyridine nucleotide-independent TR [The sequence analysis of the genome of ox motif A, which ox motif A. A compndent TR  and has ndent TR . The MjTM. mazei contains two Trxs, herein named MmTrx1 (MM_RS02345) and MmTrx2 (MM_RS05160)  B. MmTrx1 homolog , suggestM. mazei Gö1 genome also identified atypical Trxs with varying active sites. NTR was placed adjacent to the gene MM_RS12205 that encoded a predicted Trx protein, herein called MmTrx3, with a WCTAC redox motif (M. acetivorans (MaTrx7) that has been biochemically demonstrated to be reduced by an NTR [M. acetivorans (MaTrx5 and MaTrx1) failed to display insulin disulfide reductase activity and are of unknown function [Methanosarcina [A sequence analysis of the ox motif B; MmTrx3y an NTR . Other aans MaTrx that hasfunction . FDRs arfunction . Apparenosarcina ,31,32.According to the protein sequence, MmNTR has a theoretical molecular weight of 35 kDa. A homogenous sample of MmNTR protein was prepared using the protocol described in the Materials and Methods section. The purified MmNTR migrated as a single band with an apparent molecular mass of 35 kDa in sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) A. The puProtein crystals of MmNTR were obtained, and the X-ray structure was solved and refined to a resolution of 2.6 Å . The X-rStaphylococcus aureus  with a root mean square deviation value of 1.7 Å  conformation of NTRs that allows for the interaction of the disulfide bridge of the CxxC motif with the isoalloxazine ring of the flavin in a conformation competent for the reduction of the disulfide [The NADPH-binding domain is positioned in an orientation with respect to the FAD-binding domain that exposes the NADPH-binding site to the bulk solvent and approaches the CxxC motif over the isulfide . The twoEscherichia coli (EcNTR) homodimers reveals a significant difference at the dimer interface . As shown in M. mazei Gö1 is similar to that of other Methanosarcina [M. acetivorans, but not of canonical Trxs. This suggests the presence of other reduction pathways in this type of archaea. The elucidation of alternative reduction systems requires further work; possibilities might include the Fe-S proteins that have been detected in the genomes of Methanosarcina, including those that have been related to the catalytic subunit of the FTR protein that is functional in plants [A genome-wide sequence analysis showed that the composition of the Trx system of osarcina , with twn plants .The high-resolution structural analyses of various bacterial NTRs show that LMW-NTRs are homodimers with two Rossmann-fold domains in each subunit . One domThe protein was crystallized in the FO conformation, with the CxxC motif forming a disulfide bridge placed over the isoalloxazine ring of the assumed FAD. It is anticipated that MmNTR undergoes a general rearrangement that leads to interdomain motion to permit the transfer of reducing equivalents from NADPH to CxxC, and subsequently to Trx.Our attempts to solve the holo-structure by X-ray crystallography have not been successful so far due to loss of the flavin cofactor during crystallization. Therefore, we performed SAXS studies to obtain structural information on the MmNTR holo-protein, concluding that the conformation of MmNTR in solution is similar to canonical LMW-NTR proteins. The differences between the crystal and the SAXS MmNTR structures might be attributed to the loss of the flavin cofactor, likely during the crystallization process.M. mazei Gö1 and the structure of apo-MmNTR obtained by X-ray crystallography and SAXS. Our results show that the structural features of the LMW-NTR family of proteins are well-conserved in the MmNTR structure. The results suggest a primary important role of FAD. The cofactor, which may have been lost during crystallization, appears to function in protein folding since its absence impacts the ability of the enzyme to form the physiological dimer interface. In future work, the three-dimensional structure of the holo-form of the enzyme should provide more accurate data on the conformational organization of the redox-active sites.We have presented a description of the thioredoxin system of